• Korean Journal of Fisheries and Aquatic Sciences
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• v.25 no.2
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• pp.79-92
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• 1992

This study was carried out to develop new techniques useful for cryopreservation, thawing and artificial insemination, and ultrastructural changes of cryopreserved spermatozoa in rainbow trout(Oncorhynchus mykiss) . Two extenders, such as Tyrode solution and Whittingham's $T_6$ solution, were used to preserve rainbow trout sperm in refrigerator $(-20,\;-40\;and\;-70^{\circ}C)$ or liquid nitrogen $%(-196^{\circ})$. Hand-stripped semen was diluted to 1:16 with two extenders, an then the semen were frozen after mixing semen and each extender containing 1M or 1.5M DMSO solution to 1:1. After 60 days cryopreserved semen was thawed in a $13^{\circ}$ water bath, and subsequently centrifugated. After centrifugation at 1,000 rpm for 5 min thawed semen was washed with extenders, and then fertilized with fresh eggs. The results obtained in these experiments were summarized as follows: After cryopreservation, over 75% of spermatozoa were appeared motile and the survival rate was high. Following cryopreservation by the addition of cryoprotectant such as DMSO, methanol and glycerol, the fertilization rate of the thawed spermatozoa appeared over $99\%$ compared with the control having $99\%$ of fertilization rate. There was no difference between the control and experimental groups such as $(-20^{\circ}C\;-40^{\circ}C\;and\;-70^{\circ}C)$ and $-196^{\circ}$ in fertilization rate. Following cryopreservation at $-196^{\circ}$ by the addition of 1M DMSO of cryoprotectant, each fertilization rate following 24 hours and hatching rate following 24 days showed $96\%$ and $8\%$ by the addition of BSA, but showed $98\%\;and\;10%$ by no addition of BSA. Following 2 months of cryopreservation by the addition of 1M DMSO of cryoprotectant, there were $10%$ of hatching rate at $-196^{\circ}\;and\;10\%\;and\;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1M methanol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C,\;and\;28\%,\;at\;-70^{\circ}C$ Following 2 months of cryopreservation by the addition of 1M glycerol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C$, and $33\%,\;at\;-70^{\circ}C$. pollowing 2 months of cryopreservation by the addition of 1.5M DMSO of cryoprotectant, there were $27\%$ of fertilization rate at $-20^{\circ}C,\;an\;36\%\;and \;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1.5M glycerol of cryoprotectant, there were $34\% \;of\;fertilization\;rate\;at\;-20^{\circ}C, \;and\;31\%\;and\;31\%,\;respectively,\;at \;-40^{\circ}C\;and\;-70^{\circ}$. Following 2 months of cryopreservation by the addition of 1.5M methanol of cryoprotectant, there were $28\%$ of fertilization rate at $-20^{\circ}C,\;and\;29\%\;and\;28\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C.$ From 10 days and 15 days following fertilization at $13^{\circ}C\;and\;10^{\circ}C$, respectively, the mortality rate of fertilized ova was markedly increased. The middle piece of spermatozoa had two set of central doublets, nine set of outer coarse fibres, and mitochondrial sheath. Spermatozoa went through morphological changes during storage, e.g. winding of flagella, detachment of the nuclear envelope and the plasma membrane from the nucleus of the sperm head. There were $1\%$ abnormal spermatozoa in fresh sperm and about $15\%$ during storage.

• Journal of Aquaculture
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• v.20 no.3
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• pp.199-202
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• 2007

We isolated a 317 bp of partial cDNA for doublesex-and mab-3-related transcription factor-1 (DMRT-1) from the testis of olive flounder, Paralichthys olivaceus using RT-PCR. Based on the multiple sequence alignment, olive flounder DMRT-1 shared relatively high sequence homology (82 to 94%) with orthologues from other teleost species such as Atlantic halibut, Hippoglossus hippoglossus, black porgy, Acanthopagrus schlegeli and rainbow trout, Oncorhynchus mykiss. DMRT-1 mRNA was predominantly expressed in the testis of olive flounder. In our investigation for the effect of testosterone treatment in vivo on induced expression of ovarian DMRT-1 transcript, mRNA levels of DMRT-1 in ovary were significantly up-regulated by testosterone treatments (0.3 or $3.0{\mu}g$ testosterone/g body weight for 12 to 36 hours) as judged by RT-PCR analysis. In overall, transcriptional stimulation of DMRT-1 during treatments was more affected by doses of testosterone than treatment durations. This result strongly suggests that the regulation of DMRT-1 be tissue- and gender-specific in olive flounder, and also provides useful baseline knowledge on the testosterone-mediated regulation in the reproductive physiology of this species.

• Journal of fish pathology
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• v.9 no.1
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• pp.21-31
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• 1996

Concerned to the Ichthyophthiriasis of aquacultural fishes, the developmental features of Ichthyophthirius multifiliis were studied in cultured Korean catfish, Silurus asotus, and rainbow trout, Oncorhynchus mykiss. In the morphological observation, the parasite developed on two kinds of parasitic and non-parasitic phases with 6 developmental stages termed phoront, trophont, protomont, tomont, tomite, and therone. The $60{\times}40-100{\times}70{\mu}m$ fusiform or spherical phoront for the invading stage has 34-38 meridional kineties and begins to develope buccal apparatus. The 80-$800{\mu}m$ spherical or amoeboid trophont for the vegetative stage has a horseshoe shape macronucleus, a inconspicuous cytostome and developmental contractile vacuoles. The 200-$800{\mu}m$ spherical protomont for the encysting stage has a inconspicuous macronucleus, abundant contractile vacuoles; and a fine gelatinous exocyst is exuded, the baccal apparatus begins to resorb. The tomont for the encysted dividing stage has a thick cyst wall, and the buccal apparatus is resorbed completely. A small 35-$50{\mu}m$ spherical tomite for each daughter cell has a cytostome end the conspicuous oral apparatus. The $25{\times}20-60{\times}40{\mu}m$ fusiform theront for the infective stage possesses a perforatorium in the anterior end, a cytostome in the mid-point respectively and has 34-38 meridional kineties. In the experiments of the reproductive, the excysted time is related to water temperature. Tomitogenesis takes 10-14 hours at $28^{\circ}C$, 12-15 hours at $26^{\circ}C$, 16-18 hours at $22^{\circ}C$, 24-28 hour at $18^{\circ}C$, 26-51 hours at $14^{\circ}C$, and 129 hours at $9^{\circ}C$ respectively.

• Journal of fish pathology
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• v.25 no.3
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• pp.257-262
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• 2012

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was evaluated to monitor infectious hematopoietic necrosis virus (IHNV) from artificially infected rainbow trout Oncorhynchus mykiss. The cumulative mortalities of fish challenged with IHNV at $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish and $10^{4.5}\;TCID_{50}$/fish were 40%, 0% and 0%, respectively. Dead fish and survivors at 16 and 28 d post-challenge in each group were employed for IHNV detection by RT-LAMP assay and virus isolation using BF-2 cells. IHNV from $10^{4.3}$ to $10^{6.8}\;TCID_{50}/ml$ was isolated from all the dead fish and also detected in all of the examined dead fish by RT-LAMP assay. In survivors at 16 d, 60% (3/5 fish, $10^{2.8}-10^{5.05}\;TCID_{50}/ml$), 20% (1/5 fish, $10^{1.05}\;TCID_{50}/ml$) and 60% (3/5 fish, $10^{1.05}-10^{4.8}\;TCID_{50}/ml$) were found to be IHNV-positive by virus isolation in fish challenged with IHNV at $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish and $10^{4.5}\;TCID_{50}$/fish, respectively, while 20% (1/5 fish), 0% (0/5 fish) and 20% (1/5 fish) were IHNV-positive by RT-LAMP assay. No IHNV was detected in the survivors at 28 d and control fish. These results indicate that the RT-LAMP assay is useful for detection of IHNV in diseased fish although it is not enough to monitor virus in IHNV-survivors.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.4
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• pp.470-475
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• 1999

This study was conducted to evaluate the utilization of animal by-product mixture (ABPM) as a dietary animal protein source of fish meal replacer, and to determine the effect of dietary chromic oxide in growing rainbow trout, Oncorhynchus mykiss. ABPM is a mixture of five anmial by-products such as meat and bone meal (MBM) feather meal (FM), squid live, powder(SLP), poultry by-product (PBP) and blood- meal (BM) at a specific weight based ratio. Diet 1 and 2 were formulated on a isonitrogenous and a isocaloric basis of $46.5\%$ crude protein and 16.7 KJ/g diet; diet 1 (WFM 100), $100\%$ of the animal protein source came from white fish meal; diet 2 (ABPM 40), $60\%$ WFM+$40\%$ ABPM as the animal protein source; diet 3 (-Cr) commercial diet without chromic oxide; diet 4 (+Cr), commercial diet with chromic oxide. After eight weeks of feeding trials, fish fed diet 2 had a significantly lower body weight gain (WG) and feed efficiency (FE) than that of fish fed the other diets (P<0.05). When comparing diet 3 with diet 4, no significant differences were found in WG and FE (P>0.05). There were no significant differences on condition factor, hematocrit level, serum phosphorus, bone phosphorus, whole body phosphorus, and bone ash among fish from all four diet groups. Fish fed diet 4 had a significantly higher whole body lipid than that of fish fed the other diets (P<0.05), These results indicated that ABPM could be used less than $40\%$ in growing rainbow trout with a sufficient period of acclimation, In addition, the $0.5\%$ of chromic oxide can be used to determine the apparent digestibility of the nutrients in the feed without any adverse effects on growth and body composition.